Epstein-Barr virus (EBV) is a lymphotropic, human herpes virus which latently infects B lymphocytes, resulting in concomitant growth transformation of infected cells. EBV in the latent state can be activated to the viral lytic cycle by an intricate cascade of events initiated at the plasma membrane level. Virus activation is central to the pathogenesis of EBV infection. Recent topics of EBV reactivation focus on AIDS patients. We have been studying the mechanisms of EBV activation in the Burkitt's lymphoma cell line, Akata. Akata cells demonstrate prompt and synchronous activation of latent EBV after crosslinking of membrane IgG (mIgG) with anti-IgG. B cells are activated by mIg-triggered, second messenger pathways which activate protein kinase C (PKC) and Ca++/calmodulin protein kinases. We found that EBV activation was induced with calcium ionophore A23187, which was synergized by PKC agonists. The antagonists of Ca++/calmodulin or of PKC blocked the EBV activation. The cAMP analogs inhibited the activation. Crosslinking of mIgG also led to rapid tyrosine phosphorylation in the cells. With respect to the role of tyrosine phosphorylation in EBV activation, we demonstrated that the tyrosine kinase activation is an essential event in EBV activation after crosslinking mIgG. The expression of BamHI Z EBV replication activator (ZEBRA), BZLF1 immediate- early (IE) gene product is sufficient to trigger the activation for the virus lytic cycle in latently infected B cells. We analyzed the events in EBV gene expression after EBV activation by immunofluorescence, Western blotting, Northern blotting and nuclear run-off assays. The regulation of EBV activation via second messenger signals could be explained by regulation at the level of transcription of EBV IE genes. Recently, we found phosphorylation of ZEBRA in Akata cells stimulated with anti-IgG. ZEBRA was phosphorylated for serine residues, but not for threonine or tyrosine. In this proposal, we will extend this study further as follows. We will: (1) Clone Akata cells to analyze the characteristics of the clones which differ in EBV induction with anti-IgG crosslinking, to test the cellular and viral factors which regulate EBV inducibility. (2) Characterize the expression of cellular oncogenes (c- fos, c-jun, etc.) and BZLF1 gene to define their roles in EBV activation. (3) Analyze the BZLF1 promoter, to identify the responsive elements in the regulation of BZLF1 gene. (4) Characterize the phosphorylation of ZEBRA on the EBV activation. These studies could be of potential therapeutic value for EBV reactivation in patients with Burkitt's lymphoma, AIDS, etc.